Non-immunochemical qualitative and quantitative determinations of compounds found in biological samples often require separation of the compound of interest from others in the sample. Once separated, the compound of interest may be detected and/or identified by measuring some property of the compound.
Since many biological molecules have low volatilities and decompose when heated, rather than vaporizing to form gas phase molecules, separation by gas chromatography (GC) is often impossible without first preparing volatile derivatives. Therefore, high performance liquid chromatography (HPLC) is often chosen for separations of non-volatile biological molecules. In HPLC, separations are made in the liquid phase and derivatization is typically unnecessary because most molecules are soluble in at least one solvent.
Nonetheless, derivatization is useful in HPLC to enhance separation of molecules and to increase sensitivity of detection of the separated molecules. For example, the analyte molecules might not possess physical properties that can be accurately measured in the presence of solvent molecules. Detection of the analyte may be improved by derivatization with reagents to form readily detectable derivatives. For example, an analyte can be derivatized with a fluorescent compound to make it readily detectable.
Coupling a chromatographic method of separation with mass spectrometric detection permits separation of complex mixtures into their components, detection of the components, and identification of the components from their mass spectra. Since mass spectrometry (MS) requires conversion of analyte molecules to gas phase ions, coupling an HPLC column to a mass spectrometer requires a means of isolating the analyte molecules from excess liquid solvent as they emerge from the column. If the solvent were introduced into the vacuum of a mass spectrometer, the pressure increase would prevent the instrument from functioning. MS also requires that isolated analyte molecules be ionized before they can be detected. Electrospray ionization (ESI) is one method for coupling the effluent of an HPLC column to a mass spectrometer. ESI functions to remove solvent from a liquid sample without losing the analytes and to ionize the analytes.
In ESI, a stream of analyte-containing solvent is passed through a narrow capillary tube, the end of which is held at a high positive or negative electrical potential. The strong electric field that surrounds the end of the capillary tube causes the emerging liquid to leave the capillary as a fine mist of droplets. The droplets acquire an excess of charge (positive or negative depending upon the potential applied to the capillary) as they leave the capillary and enter an atmospheric pressure evaporation chamber (ESI is an example of an atmospheric pressure ionization, or API, method). As solvent continues to evaporate from the droplet the charge density in each droplet continues to increase. Eventually, repulsion between ions in the droplet exceeds the surface tension of the droplet and ions are expelled into the gas phase in a process termed ion evaporation. Before the analyte ions formed in the evaporation chamber are selectively introduced into the mass spectrometer, they collide with other ions and neutral molecules. During these collisions, charges may be transferred between species to form new ions and new neutral molecules in a process called chemical ionization.
In positive ion mode ESI (i.e. the capillary is held at a high positive potential), an important chemical ionization process is transfer of protons (H+) between species in the evaporation chamber. Positive ion mode ESI is widely used for vaporizing and ionizing biological molecules because biological molecules typically have multiple sites in their structures that have an affinity for protons. Proteins, in fact, can attract and hold enough protons and other cations (e.g. Na+) during the ESI process that they can form multiply charged ions. Smaller biological molecules, however, may not ionize efficiently, especially if they do not possess groups of atoms having an affinity for protons. Derivatization of such molecules offers one way to improve the efficiency of ionization in ESI and hence detection by MS.
In combined HPLC-ESI-MS, both separability in the HPLC column and detection by ESI-MS determine whether the method may be used to determine particular analytes. Derivatization to improve separation by HPLC can have a detrimental effect on detection by ESI-MS, and the converse is true, making it difficult to find appropriate derivatization schemes for HPLC-EIS-MS.